Ferritin in glia is required for NB maintenance and proliferation

(A) NB number and proliferation rate after Fer1HCH or Fer2LCH RNAi in glia.

(B) Quantification of NB number in CB (A).

(C) Quantification of NB proliferation rate in CB (A).

(D and E) RT-PCR analysis of Fer1HCH and Fer2LCH mRNA levels (D) and normalized quantification (E) after glial ferritin knockdown.

(Fand H) Western blot of Fer1HCH (F) and Fer2LCH (H) after ferritin knockdown in glia. Tubulin was used as a loading control.

(G and I) Quantification of normalized Fer1HCH intensity in (G) and Fer2LCH intensity in (I).

(J) Rescue of NB number and proliferation when simultaneously overexpressing RNAi-resistant

Fer1HCH and Fer1HCH RNAi (or Fer2LCH and Fer2LCH RNAi) in glia. (K and L) Quantification of NB number and proliferation rate in (J).

(B, n=11; C, n=10; E, G and I, n=3; K, n=8,8,9,7,9; L, n=9,8,8,7,12; ns, not significant; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; Dpn, NB nuclei, red; PH3, green; DAPI, nuclei, blue).

The phenotype of ferritin knockdown in different glial subpopulations.

BDSC: Bloomington Drosophila Stock Center; DGRC: Drosophila Genetic Resource Center; +++: strong phenotype; ++: weak phenotype; -: no phenotype.

Ferritin is produced mainly in glial cells in the Drosophila CNS and secreted into NBs through a vesicle dependent pathway

(A) The pattern of Dsred Stinger driven by Fer1HCH/Fer2LCH-GAL4 in CNS.

(B) The distribution of ferritin labeled by Fer1HCHG188 in CNS of control and glial ferritin knockdown.

(C) Overexpressed ferritin tagged with GFP and mCherry in glia secreted into NBs.

(D) The distribution of Fer1HCHG188 when blocking glial vesicular trafficking via overexpressing dominant-negative dynamin.

(E) Quantification of (D), GFP signal in NBs when overexpressing shi ts in glia. (E, n=50, 48; **: p<0.01)

Glial ferritin defects lead to iron deficiency in NBs

(A) The conditions of brain development in Drosophila with feeding iron chelator BPS or supplementary iron FAC.

(B) Iron depletion by adding DFP to food exacerbated the phenotype of NB loss induced by glial ferritin knockdown.

(C) Quantification of NB number in (B).

(D) Zip13 in CNS is produced mainly by glial cells, but not NBs.

(E) Brain defects in Zip13 knockdown or double knockdown of Zip13 and Fer2LCH. (F and G) Quantification of NB number and proliferation rate in (E).

(H) Iron supplement can rescue the NB proliferation in Zip13 RNAi driven by repo-GAL4.

(I) Quantification of NB proliferation in (H).

(C, n=6; F, n=6,10,11,8; G, n=13,10,10,5; I, n=8; ns, not significant; *: p<0.05; **: p<0.01; ****: p<0.0001)

Glial ferritin defects result in impaired Fe-S cluster activity and ATP production

(A) The determination of cytosolic aconitase activity.

(B) Knockdown of CIA complex in NBs.

(C) Quantification of NB proliferation in (B).

(D) The schematic diagram of NB sorting procedure.

(E) Venn diagram of downregulated genes after glial ferritin knockdown.

(F) GO enrichment of down-regulated genes in glial ferritin knockdown comparing with control.

(G) KEGG pathway enrichment of down-regulated genes after glial ferritin knockdown.

(H) NAD+ level was indicated by SoNar.

(I) Quantification of normalized SoNar signal in (H).

(J) The determination of ATP level in CNS.

(K) NB number in glial ferritin knockdown was rescued by Ndi1 overexpression in NBs.

(L) Quantification of NB number in (K).

(A and J, n=3; C, n=5,6,6,9; I, n=37,42; L, n=7,7,6,6,7; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001)

Glial ferritin defects lead to the premature differentiation of NBs

(A) Temporal window of glial ferritin maintaining NBs. The temperature shift of flies with glial ferritin knockdown from the permissive temperature (18℃) to the restrictive temperature (29℃).

(B) Quantification of NB number and proliferation in (A).

(C) Pros staining of knocking down glial ferritin.

(D) Quantification of ratio of Pros+Dpn+ NBs to Dpn+ NBs.

(E) Knockdown of pros in NBs rescues the NB number.

(F) The ratio of brain with NB loss in (E).

(B, n=6, 5, 6; n=6, 5, 4; D, n=5; F, n=3; *: p<0.05; ***: p<0.001; ****: p<0.0001)

Ferritin functions as a potential target for tumor suppression

(A) Knockdown of ferritin in glia could inhibit the tumor induced by brat RNAi or numb RNAi.

(B) Quantification of tumor size in (A).

(C) Iron chelator BPS supplemented in the food suppressed brain tumor growth.

(D) Quantification of tumor size in (C).

(E) Bioluminescence signal in mice with glioma on day 14.

(F) Quantification of bioluminescence signal in (E).

(G) Kaplan-Meier survival curve of mice treated with DFP.

(B, n=10,7,7,5; D, n=8,6,6,6; F, n=6,7; G, n=7,6; *: p<0.05; ***: p<0.001; ****: p<0.0001)

The level of ferritin is controlled by NB proliferation

(A) Fer1HCH subunit level in Drosophila CNS after manipulating proliferation.

(B) Fer2LCH subunit level in Drosophila CNS after manipulating proliferation.

(C) Quantification of Fer1HCH level in (A).

(D) Quantification of Fer2LCH level in (B).

(E) The model of glial ferritin regulating NBs.

(C and D, n=3; ns, not significant; *: p<0.05; **: p<0.01; ***: p<0.001)

Glial ferritin knockdown leads to low NB proliferation and number

(A) Quantification of NB number in VNC.

(B) Quantification of NB proliferation rate in VNC.

(C) EdU incorporation in CNS after knocking down glial ferritin.

(D) Knocking down Fer2LCH by nrv2-GAL4 led to similar but weaker phenotype in brain in comparison with pan-glial ferritin knockdown.

(A, n=11; B, n=10; ****: p<0.0001)

Verify the phenotype induced by glial ferritin knockdown using different manipulations

(A) NB number and proliferation in other RNAi lines targeting on ferritin.

(B) Rescue of NB number in VNC when simultaneously overexpressed RNAi-resistant Fer1HCH and Fer1HCH RNAi (or Fer2LCH and Fer2LCH RNAi) in glia.

(C) Rescue of NB proliferation rate in VNC when simultaneously overexpressed RNAi-resistant Fer1HCH and Fer1HCH RNAi (or Fer2LCH and Fer2LCH RNAi) in glia.

(D) NB number and proliferation in Fer1HCHDN. (B, n=8,7,9,7,8; C, n=9,8,8,7,12; ****: p<0.0001)

Brain defects induced by glial ferritin knockdown were caused by iron deficiency

(A) Iron deficiency by blocking iron absorption in gut also induced similar brain defects caused by BPS addition.

(B) Quantification of NB number in (A).

(C) Quantification of NB proliferation in (A).

(D) Brain development of glial ferritin knockdown after feeding FAC.

(E) The ratio of brain with NB loss after adding FAC.

(F) NB number and proliferation of Zip13 mutant.

(G and H) Quantification of NB number and proliferation rate in (F).

(B, n=10; C, n=5; E, n=3; G, n=10,9; H, n=9,8; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001)

ROS accumulates in CNS after glial ferritin knockdown, but inhibiting ROS cannot restore NB number and proliferation

(A) ROS level marked by Gstd-GFP after knocking down ferritin in glia.

(B) Quantification of ROS level in (A).

(C) Overexpress antioxidant sod1 or cat in glial cells.

(D) Overexpress antioxidant sod1 or cat in NBs.

(E) Mitochondrial morphology by TEM upon glial ferritin knockdown.

(F) Quantification of normal mitochondria in (E). Intact mitochondria with their cristae visible were considered normal.

(B, n=5,4,5; F, n=23,27,28; ****: p<0.0001; ns, not significant)

Glial ferritin knockdown does not induce apoptosis in the CNS

(A) Apoptotic signal labeled by Caspase-3 of glial ferritin knockdown.

(B) Quantification of Caspase-3 negative NB ratio.

(C) TUNEL staining of Fer1HCH RNAi driven by repo-GAL4.

(D) Quantification of TUNEL positive cell per 1×105 μm2.

(E) P35 overexpression in glia could not rescue brain defects of ferritin knockdown.

(F) Glial morphology was intact when knocking down glial ferritin. (B, n=5,4,4; D, n=3; ns, not significant)

Validation of RNA-seq data by qRT-PCR using sorted NB lineages

(A) qRT-PCR analysis of ND-15 and CG15715 to validate the enrichment result. (n=3; *: p<0.05; **: p<0.01)

NB loss upon glial ferritin knockdown is not due to NB origin and apoptosis

(A) Temporal window of glial ferritin maintaining NBs. The temperature shift of flies with glial ferritin knockdown from the restrictive temperature (29℃) to the permissive temperature (18℃).

(B) Quantification of NB proliferation in (A).

(C) The CNS of the first-instar larvae with glial ferritin knockdown.

(D) P35 overexpression in NBs could not rescue the brain defects induced by glial ferritin knockdown.

(B, n=5; ns, not significant)

Glial ferritin defects do not affect NB reactivation

(A) NB size upon glial ferritin knockdown was not changed.

(B) Activation of Insulin signaling could not restore NB number and proliferation.

(C) Quantification of NB proliferation in (B). (A, n=100; C, n=8, 5; ns, not significant)

Iron chelator DFP inhibits glioma progression in mice

(A) The appearance and HE staining of brains in mice with DFP injection on day 7, 14 and 21. Scar bar: 1000 μm.

(B and C) Quantification of HE staining in murine brains on day 14 (B) and 21 (C). (B, n=6; C, n=9,8; ***: p<0.001; ****: p<0.0001.)